Cell culture media for enhanced protein production

ABSTRACT

A cell culture medium is provided which constrains cell growth and enhances antibody production. The high glucose medium of the invention is preferably saturated at 40° C. with essential amino acids.

FIELD OF THE INVENTION

[0001] This invention relates to cell culture media, which improvesprotein production, constrains cell growth and extends cell longevity invitro culture, and to methods for the production and use of such media.

BACKGROUND OF THE INVENTION

[0002] The increasing demand for monoclonal antibodies (MABs) useful inresearch, diagnosis, therapy and purification purposes has created aneed to optimize production techniques. The prior art includes improvedbioreactor designs and bioreactor operation to increase cell densitiesor the longevity of the culture by nutrient feedings.

[0003] Bioreactors have been operated in fed-batch, immobilized,perfusion and continuous modes. Alternate strategies, such as the use oftemperature, media formulation, including the addition of mouseperitoneal factors, growth inhibitors, autocrine factors or cyclicmononucleotides and hyperstimulation by osmolarity stress, have beenused to enhance protein production. These approaches have shown onlymarginal success.

[0004] Commonly used basal cell culture media are RPMI 1640, DMEM(Dulbecco's modified Eagle's medium), Ham's F12 and DMEM/F12 (DF).Murakami (1989) (1)¹ describes a modified medium, eRDF, prepared fromRDF(RPMI:DMEM: F12=2:1:1) by

[0005] Oh, et al. (1995) (4) reports that hybridomas increased metabolicactivities and amino acids uptake via the Na+ dependent symports tocompensate for the osmotically elevated external environment.

[0006] Oh, et al. (1996) (5) describes the application of flow cytometryin examining the relationships between total cellular monoclonalantibody content, cell size, and cell cycle distribution of hybridomassubjected to environmental stress.

SUMMARY OF THE INVENTION

[0007] The invention provides cell nutrient media which enhance proteinproduction and prolong in vitro cell viability. The method cell cultureutilizing such media are an important aspect of the invention.

[0008] The media and the methods of the invention are applicable to theculture of cells of any type in bioreactors of all kinds.

DETAILED DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1—growth of hybridomas 2HG11 and TBC3 in BTC-28101 andcontrol DMEM/F12 media.

[0010]FIGS. 2a-2 d reflect the result of hollow fiber bioreactorexperiments in which BTC-28101 was utilized.

[0011]FIG. 2a represents levels of antibody produced.

[0012]FIG. 2b sets forth medium pH data.

[0013]FIG. 2c reports glucose utilization.

[0014]FIG. 2d reports cell viability.

[0015]FIG. 3a—growth of hybridoma 2HG11 in serum-free BTC-28101 andcommercial media Hb and PFHM available from Gibco.

[0016]FIG. 3b—IgG concentration in serum-free BTC-28101 and commercialmedia Hb and PFHM available from Gibco.

[0017]FIG. 4—growth of hybridomas 2HG11 and TBC3 in BTC-28102 andcontrol DMEM/F12 media.

[0018]FIG. 5—growth of CHO cells in BTC-28103 and control IMDM media.IMDM refers to Iscove's Modified Dulbecco's Media.

[0019]FIG. 6 presents a correlation of percent dry weight of amino acidin the media components with MAB production in ug/ml. The figureillustrates an unexpected increase in MAB concentration when the percentamino acid content in the media exceeds about 20%. In the Figure D/Frefers to Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, 1:1Mixture.

DEFINITIONS

[0020] Cell Culture Medium—Any medium in which cells of any type may becultured.

[0021] Bioreactor—Any device in which cells may be cultured. Includesstationary flasks, spinner flasks and hollow fiber bioreactors.

[0022] Basal Medium—A cell culture medium that contains all of theingredients essential to cell metabolism, e.g., amino acids, lipids,carbohydrates, vitamins and mineral salts RPMI, DMEM, Ham's 12, and RDFare examples of basal media.

[0023] Essential Amino Acids—Arg, Cys, Gln, His, Pro, Ile, Leu, Lys,Met, Phe, Thr, Trp, Tyr and Val.

[0024] Non-Essential Amino Acids—Ala, Asn, Asp, Gln, Gly, Ser.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The invention provides a method for improving protein productionin cultures of protein producing cells. In particular, the inventioncomprises culturing hybridomas antibody producing cells in a highosmolarity aqueous medium comprising a high concentration of aminoacids, in particular the essential amino acids, and an energy sourcesuch as glucose or sucrose. The medium is substantially saturated ataround 40° C. with an amino acid or acids essential to the metabolism ofthe culture cells. The medium of the invention may contain 5.50 to 20grams per liter of total or gross amino acids in solution or suspensionand 5.50 to 20 grams per liter of a carbohydrate energy source,preferably glucose, in solution. The gross amino acids comprise at least20%, preferably, from about 25% to about 50% of the total dry weight ofthe medium components. Cells may appropriately be adapted to the highosmolarity media of this invention by passaging.

[0026] The osmolarity of the medium is from 320 to 450. Sodium chlorideis the preferred osmolyte.

[0027] The media and the methods of the invention are useful in allforms of bioreactors. The benefits of the invention are realized instatic, batch, shaker flask, and spinner flask and hollow fiberbioreactor culture procedures.

[0028] Cells of all kinds may be cultured in any of the methods of theinvention. Culture of recombinant protein expressing mammalian cells,e.g., CHO cells, is an important aspect of the invention. Many types ofmammalian cells, which contain recombinant protein containing expressionvectors are known. See, e.g., Acklin, C., et al. (Recombinant humanbrain-derived neurotrophic factor (ΓHuBDNF). Disulfide structure andcharacterization of BDNF expressed in CHO cells) Int. J. Pept. ProteinRes. (1993) 41:548-52; Fukushima, K., et al., (N-linked sugar chainstructure of recombinant human lymphotoxin produced by CHO cells; thefunctional role of carbohydrate as to its lectin-like character andclearance velocity) ABB (1993) 304:144-53; Hayakawa, T., et al. (In vivobiological activities of recombinant human erythropoietin analogsproduced by CHO cells, BHK cells and C127 cells) Biologicals (1992)20:253-7; Israel, D. I., et al. (Expression and characterization of bonemorphogenetic protein-2 in Chinese hamster ovary cells) GF (1992)7:139-50; Langley, K. E., et al. (Purification and characterization ofsoluble forms of human and rat stem cell factor recombinantly expressedby Escherichia coli and by Chinese hamster ovary cells) ABB (1992)295:21-8; Lu, H. S., et al. (Post-translational processing ofmembrane-associated recombinant human stem cell factor expressed inChinese hamster ovary cells) 298:150-8; Malik, N., et al.,(Amplification and expression of heterologous oncostatin M in Chinesehamster ovary cells) DNA Cell Biol. (1992) 11:453-9; Nagao, M., et al.(Production and ligand-binding characteristics of the soluble form ofmurine erythropoietin receptor) Biochem. Biophys. Res. Commun. (1992)188:888-97; Rice, K. G., et. al. (Quantitative mapping of the n-linkedsialyloligosaccharides of recombinant erythropoietin; combination ofdirect high-performance anion-exchange chromatography and2-aminopyridine derivatization) Anal. Biochem. (1992) 206:278-87;Schmelzer, C. H., et al. (Purification and partial characterization ofrecombinant human differentiation-stimulating factor) Protein Expr.Purif. (1990) 1:54-62; Schmelzer, C. H., et al. (Biochemicalcharacterization of human nerve growth factor) J. Neurochem. (1992)59:1675-83; Sima, N., et al. (Tumor cytotoxic factor/hepatocyte growthfactor from human fibroblasts; cloning of its cDNA, purification andcharacterization of recombinant protein) Biochem. Biophys. Res. Commun.(1992) 180:1151-8; Sun, X. J., et al. (Expression and function of IRS-1in insulin signal transmission) J. Biol Chem (1992) 267:22662-72;Suzuki, A., et al. (Biochemical properties of amphibian bonemorphogenetic protein-4 expressed in CHO cells) BJ (1993) 291:413-7;Tressel, T. J., et al. (Purification and characterization of humanrecombinant insulin-like growth factor binding protein 3 expressed inChinese hamster ovary cells) Biochem. Biophys. Res. Commun. (1991)178:625-33. See also Lucas, B. K., et al. (High-level production ofrecombinant proteins in CHO cells using a dicistronic DHFR intronexpression vector) (1996) Nucleic Acids Res. 24: 1774-9.

EXAMPLE 1 Medium BTC 28101

[0029] The dry powder form of Medium BTC-28101 was prepared as twoseparate components (A) and (B) as listed in Table I. The ingredientswere milled to fine dry powder prior to use. To prepare the medium,Component (A) was dissolved in 90% by volume of pyrogen-free water. Themixture was warmed to around 40° C. and stirred for one hour to fullydissolve the powder, and then cooled down to room temperature. Component(B) was added and stirred another hour to dissolve. pH was adjusted to7.0 by addition of NaOH. Water was added to make up to the desiredvolume. The osmolarity of the medium was in the range of 330-335mOsm/Kg. TABLE I Composition of Medium BTC-28101 in mg/L Component (A)Amino Acids Alanine 13.4 Arginine.HCl 1,162.9 Asparagine.H2O 189.2Aspartic acid 80.0 Cystine.2HCl 105.4 Cysteine.HCl.H2O 105.4 Glutamicacid 79.4 Glutamine 1,997.2 Glycine 85.6 Histidine.HCl.H2O 150.9Hydroxyproline 63.0 Isoleucine 314.8 Leucine 330.6 Lysine.HCl 394.6Methionine 98.4 Phenylalanine 148.6 Proline 110.6 Serine 170.2 Threonine221.6 Tryptophan 36.8 Tyrosine 174.0 Valine 218.0 Component (B) MineralSalts CaCl2 (anh) 82.1 CuSO4.5H2O 0.00075 FeSO4.7H2O 0.220 KCl 372.8MgSO4 (anh.) 52.4 NaCl 6,136.2 Na2HPO4 (anh.) 484.1 ZnSO4.7H2O 0.230Vitamins Biotin 0.102 D-Ca pantothenate 1.240 Folic acid 8.800Putrescine.2HCl 0.040 Niacinamide 1.510 Para-aminobenzoic 0.510 acidPyridoxine.HCl 0.520 Pyridoxal.HCl 1.000 Riboflavin 0.210 Thiamine.HCl1.585 Vitamin B12 0.342 Carbohydrates and derivatives D-glucose 6,846.0Na Pyruvate 110.0 Nucleic acid derivatives Thymidine 5.7 Hypoxanthine1.0 Lipids and derivatives Choline bitartrate 55.7 i-inositiol 104.5Linoleic acid 0.020 Lipoic acid 0.050 Thiol compound Glutathione(reduced) 0.490 Buffers HEPES 3,570.0 NaHCO3 1,130.0 PH indicator Phenolred 6.0 The composition of the medium BTC-28101 is: Glucose (mg/l) 6.846Amino acids (mg/l) 6.251 Amino acids (% d.w.*) 24.8 *dry weight of mediaingredients

EXAMPLE 2 Effect of Medium BTC-28101 on IgG Production in Hybridomas inShake Flask Culture

[0030] This example compares cell growth and monoclonal antibodyproduction in two hybridoma cell lines 2H11 (antihuman chronicgonadotropin) and TBC3 (antihuman IgG) in the serum supplementedBTC-28101 medium of Example 1 versus DMEM/F12 (Dulbecco's modifiedEagle's medium; Ham's F12.1-1).

[0031] The experiment was set up in shaker flasks with 100 ml mediasupplemented with 10% FBS (Fetal Bovine Serum). Inoculum cells wereadapted and maintained by daily passaging at 2×10⁵/ml with therespective fresh medium for at least a week, and the viability of eachinoculum culture was above 90% before use. Batch culture was started byinoculating at 2×10⁵/ml into the respective medium. Samples were takendaily to follow the cell growth by trypan blue staining andhemocytometer counting. Monoclonal antibody concentration in the culturesupernatant was determined by ELISA analysis. The effect on cell growthis shown in FIG. 1. Maximum concentration of Ig at the end of thecultures are summarized in Table II: TABLE II Maximum Ig Concentrationin the Cultures with BTC-28101 and Control DMEM/F12 Media Max IgConcentration (ug/ml) Cell Line DMEM/F12 BTC-28101 2HG11 50 270 TBC3 84450

EXAMPLE 3

[0032] Hybridoma cell line TH12 (anti-theophylline) was cultured ineither the BTC-28101 media of Example 1 or a DMEM formulation. Cellswere inoculated into 100 ml of BTC-28101 or control medium DMEM at2×10⁵/ml in 250 ml spinner flasks, both media were supplemented with 10%FBS. Similar procedure as stated in Example 2 was followed for preparingthe inoculum cultures, and for monitoring the batch. TH12 producedhigher concentrations of antibody in BTC-28101 than in the formulationof DMEM. As Table III shows, cell numbers and cell viability were alsohigher in 28101. TABLE III TH12 Batch Culture:Cell Counts andViabilities DATE MEDIUM CELL COUNT VIABILITY D0 BTC-28101   2 × 10⁵/ml100% 1/30/95 DMEM   2 × 10⁵/ml  90% D1 BTC-28101 5.6 × 10⁵/ml 100%1/31/95 DMEM 2.6 × 10⁵/ml 100% D2 BTC-28101 2.4 × 10⁶/ml  98% 2/1/95DMEM 0.8 × 10⁶/ml  91% D3 BTC-28101   3 × 10⁶/ml  98% 2/2/95 DMEM 2.2 ×10⁶/ml  97% D4 BTC-28101 3.4 × 10⁶/ml  93% 2/3/95 DMEM 1.4 × 10⁶/ml  77%D5 BTC-28101 3.8 × 10⁶/ml  69% 2/4/95 DMEM 0.5 × 10⁶/ml  32% D6BTC-28101 8.4 × 10⁵/ml  25% 2/5/95 DMEM 3.6 × 10⁵/ml  20% D7 BTC-281015% day 7 2/6/95 DMEM only

[0033] Table IV demonstrates enhanced Ig production and specificantibody titer when BTC-28101 is used. TABLE IV TH12 Batch Culture: IgConcentrations and Specific Antibody Titers. DATE MEDIUM Ig mg/Ml¹TITER² D1 BTC-28101 539 ug/ml 1,600 1/31/95 DMEM 447 ug/ml   400 D2BTC-28101 468 ug/ml 12,800 2/1/95 DMEM 397 ug/ml   3,200 D3 BTC-28101681 ug/ml 12,800 2/2/95 DMEM 440 ug/ml   6,400 D4 BTC-28101 752 ug/ml  6,400 2/3/95 DMEM 518 ug/ml   3,200 D5 BTC-28101 823 ug/ml 25,6002/4/95 DMEM 553 ug/ml   6,400 D6 BTC-28101 1,500 ug/ml 25,600 2/5/95DMEM   489 ug/ml   6,400 D7 BTC-28101 1,190 ug/ml 25,600 2/6/95 DMEM  560 ug/ml   3,200 # and reading optical densities at 280 nm. # solidphase.

EXAMPLE 4

[0034] Hybridoma cell line DI16 (anti-Dirofilaria immitis) was culturedin either BTC-28101 or DMEM. DI16 produced higher concentrations ofantibody in BTC-28101 than in the in-house formulation of DMEM. Table Vshows that cell numbers and cell viability were also higher inBTC-28101. TABLE V DI16 Batch Culture:Cell Counts and Viabilities DATEMEDIUM CELL COUNT VIABILITY D0 BTC-28101   2 × 10⁵/ml 98% 3/31/95 DMEM  2 × 10⁵/ml 98% D3 BTC-28101 7.2 × 10⁵/ml 83% 4/03/95 DMEM 8.4 × 10⁵/ml99% D4 BTC-28101 5.4 × 10⁵/ml 57% 4/04/95 DMEM 1.5 × 10⁵/ml 26% D5BTC-28101   6 × 10⁵/ml 58% 4/05/95 DMEM 0.5 × 10⁵/ml  8% D6 BTC-281013.9 × 10⁵/ml 30% 4/06/95 DMEM —  0% D7 BTC-28101 3.5 × 10⁵/ml 21%4/07/95 DMEM —  0% D8 BTC-28101 1.8 × 10⁵/ml 10% 4/08/95 DMEM —  0%

[0035] Table VI reports comparative Ig titers and concentration. TABLEVI D116 Batch Culture: Ig Titers and Concentrations DATE MEDIUM IgTITER¹ Ig mg/ml² D3 BTC-28101 1:1,024 1.09 4/03/95 DMEM 1:256 0.770 D4BTC-28101 1:2,048 0.882 4/04/95 DMEM 1:512 0.926 D5 BTC-28101 1:2,0480.940 4/05/95 DMEM 1:512 0.654 D6 BTC-28101 1:2,048 1.28 4/06/95 DMEM1:512 0.746 D7 BTC-28101 1:2,048 1.11 4/07/95 DMEM Culture Cultureterminated terminated D8 BTC-28101 1:2,048 1.39 4/08/95 DMEM CultureCulture terminated terminated # and reading optical densities at 280 nm;mg/ml = O.D. 280 × dilution factor ÷ 1.41 # extinction coefficient.

EXAMPLE 5

[0036] Hybridoma cell line NP11 (anti-N-acetylprocainamide) was culturedin either BTC-28101 or DMEM. This cell line was slightly slower than theother cell lines to respond to BTC-28101 with enhanced levels ofantibody production; variations for different cell lines are notsurprising. It is significant that the BTC-28101 culture producedsubstantial levels of antibody when cultures in DMEM were no longerviable. The ability to keep cultures producing for longer periods oftime is a significant advantage of BTC-28101. See Table VII. TABLE VIINP11 Batch Culture:Cell Counts and Viabilities DATE MEDIUM CELL COUNTVIABILITY D0 BTC-28101   2 × 10⁵/ml 100% 4/07/95 DMEM   2 × 10⁵/ml 100%D1 BTC-28101  1.4 × 10⁵/ml 100% 4/08/95 DMEM  2.3 × 10⁵/ml 100% D3BTC-28101   8 × 10⁵/ml  98% 4/10/95 DMEM 1.04 × 10⁶/ml  84% D4 BTC-28101 8.4 × 10⁵/ml  81% 4/11/95 DMEM  6.7 × 10⁵/ml  55% D5 BTC-28101  1.2 ×10⁶/ml  72% 4/12/95 DMEM  2.9 × 10⁵/ml  21% D6 BTC-28101  8.4 × 10⁵/ml 52% 4/13/95 DMEM   1 × 10⁵/ml  0% D7 BTC-28101  7.6 × 10⁵/ml  41%4/14/95 DMEM —  0% D10 BTC-28101  1.3 × 10⁵/ml  5% 4/17/95 DMEM —  0%

EXAMPLE 6 Effect of BTC-28101 on IgG Production in Hollow Fiber Culture

[0037] Performance of the hybridoma in a “mini” hollow fiber bioreactor(UniSyn Technologies, Inc.'s “Mini Mouse” bioreactor) supplied with FBSsupplemented BTC-28101 was compared with the control FBS supplementedDMEM. The results are shown in Table VIII. Comparable levels of antibodywere produced by this hybridoma in the control DMEM and in BTC-28101.However, the control culture was terminated after day 13 when viabilitywas <10%. In contrary, the cells in BTC-28101 remained highly viable,and the culture was terminated only because of shortage of mediumsupply. TABLE VIII Comparison of Ig Titer of Hollow Fiber Culture inBTC-28101 and Control DMEM Media Time (day) BTC-28101 DMEM  2 1:6,4001:51,200  5 1:102,400 1:204,800  7 1:204,800 1:204,800  9 1:204,8001:102,400 12 1:204,800 1:102,400 14 1:204,800 — 16 1:102,400 — 191:51,200 —

EXAMPLE 7 Hollow Fiber Bioreactors—28101 Medium

[0038] This hollow fiber bioreactor experiment involved a particularcell line which normally produces a few hundred ug/ml of MAB inconventional method. FIG. 2a indicates that this cell line performedsubstantially better in BTC-28101. Data for pH of the medium (FIG. 2b),glucose utilization (FIG. 2c), and cell viability (FIG. 2d) arepresented. Cells growing in BTC-28101 in hollow fiber bioreactors do notappear to utilize glucose from the medium at the rate normally seen withconventional media. Monitoring of glucose utilization is a standardmeans of monitoring the progress of cells in hollow fiberbioreactors—the higher the level of glucose utilization, the better thecells are growing.

EXAMPLE 8 Spinner Flask Experiments with BTC 28101 Medium

[0039] Anti-theophylline hybridoma cells were inoculated into 250 ml ofDifco's preparation of BTC 28101 or DMEM at 2×10⁵ cells/ml in 500 mlspinner flasks. Both media were supplemented with 10% FBS, 2%L-glutamine, and 1% pen-strep. Five ml samples were collected from eachflask on the days indicated. Cell viability was determined each daysamples were collected, and antibody concentrations were determined forall samples by radial immunodiffusion (RID) after all had beencollected. Until that time, the samples (with cell material removed bycentrifugation) were stored at −20° C. Antibody concentrations, asdetermined by RID, and cell viabilities are described in Table IX: TABLEIX μg/ml Ab Cell Viability Day DMEM Difco DMEM Difco 0  <125* <125 95%95% 1 <125 <125   94% 95% 3 <125 <125   95% 95% 4 <125 <125   58% 86% 7<125 176 0 27% 8 562 0 21% 9 473 0 23% 14  1,035   0 19%

[0040] It is significant that the cell line utilized produced 1 mg/mlunder conditions described. Specifically, the spinner flasks did notprovide ideal culture conditions. Once the experiment was set up, themedium was never replaced or replenished. Consequently, metabolites anddead cells continued to accumulate.

EXAMPLE 9

[0041] Anti-theophylline hybridoma cells were inoculated into 100 ml ofDifco's preparation of BTC 281010 or in-house medium (DMEM) at 2×10⁵cells/ml in 250 ml spinner flasks. All other parameters were asdescribed for Example 8. Antibody concentrations, as determined by RID,and cell viabilities are described in Table X: TABLE X μg/ml Ab CellViability Day DMEM Difco DMEM Difco 1  <125* <125   95% 99% 3 <125 15687% 99% 4 <125 209 49% 74% 5 <125 436 16% 65% 6 <125 417  9% 39% 7 <125417 0 14% 9 400 0 10%

[0042] Note that the culture volume in Example 9 was one-half that inExample 8. Consequently, nutrients may have depleted more quickly andmetabolites or other materials accumulated in inhibitory concentrationsmore rapidly.

EXAMPLE 10 BTC-28101 on IgG Production in Serum-Free Culture

[0043] This example compares cell growth and monoclonal antibodyproduction in a hybridoma cell line (2HG11) in serum-free BTC-28101 andother commercially available serum-free media available from Gibco.

[0044] Hybridoma 2HG11 has been adapted to serum-free conditions in therespective media. All media were supplemented with insulin, transferrin,ethanolamine and selenite. Cells were inoculated into 100 ml ofBTC-28101 or control media at 2×10⁵/ml in 250 ml shaker flasks. Theresults on growth and IgG production are shown in FIGS. 3a and 3 b.

EXAMPLE 11 Preparation and Use of BTC-28102 to Culture Hybridomas

[0045] Nutrient contents of the Medium BTC-28101 was further enhanced toformulate Medium-28102. To prepare this medium, Component (C) wasprepared according to the composition in Table XI and milled to dry finepowder. The powder was sterilized by gamma-irradiation and added to 100ml of BTC-28101, constituting the Medium BTC-28102. Osmolarity of themedium was around 400 mOsm/Kg. TABLE XI Composition of Supplement toMedium BTC-28101 to Make Up BTC-28102 Component (C) in mg Alanine 2.0Arginine.HCl 174.4 Asparagine.H2O 28.4 Aspartic acid 12.0 Cystine.2HCl31.6 Glutamic acid 11.9 Glutamine 299.6 Glycine 12.8 Histidine.HCl.H2O22.6 Hydroxyproline 9.5 Isoleucine 47.2 Leucine 49.6 Lysine.HCl 59.2Methionine 14.8 Phenylalanine 22.3 Proline 16.6 Serine 25.5 Threonine33.2 Tryptophan 5.5 Tyrosine 26.1 Valine 32.7 Glucose 342.3

[0046] Cystine is utilized in lieu of cysteine which is toxic to cellsat high concentration. The composition of medium BTC-28102 is: Glucose10.269 g/l Amino Acids 15.628 g/l Amino Acids ( % d.w. of 41.1 mediaingredients)

[0047] Inoculum cells were adapted to Medium BTC-28101 following theprotocol stated in Example 2 and inoculated at 2×10⁵/ml when startingthe shaker batch, along with the control cells in 100 ml of DMEM/F12medium. The effects on cell growth are shown in FIG. 4. Maximumconcentration of Ig in the culture are summarized in Table XII. TABLEXII Maximum Ig Concentration in the cultures with BTC-28102 and ControlDMEM/F12 Media Max Ig Concentration (μg/ml) Cell Line DMEM/F12 BTC-281022HG11 50  490 TBC3 84 1200

EXAMPLE 12 Preparation and Use of BTC-28103 to Culture CHO Cells

[0048] This invention illustrates use of the invention to culturemammalian cells that express natural or recombinant protein. BTC-28103was prepared as in BTC-28101 but the buffer contents of HEPES and NaHCOwere increased to 8330 mg/l and 2650 mg/l, respectively. As a result,osmolarity of the medium was increased to 360 mOsm/Kg. CHO (Chinesehamster ovary) cells were adapted to grow in suspension and cultured in100 ml of BTC-28103 and the control IMDM in shaker flasks, both suppliedwith 10% FBS, thymidine and hypoxanthine. Growth of the cultures wasfollowed daily by hemocytometer counting and presented in FIG. 5.

[0049] The media of the invention is useful to culture proteinexpressing cell lines in the various forms of available bioreactors. Inparticular, media of this invention may be used as the intracapillarymedium in hollow fiber bioreactor culture of recombinant proteinexpressing CHO cells.

EXAMPLE 13

[0050] Table XIII indicates the composition of the commerciallyavailable media RPMI, D/F and eRDF and of the 28101 (Example 1) and28102 (Example 11) media of the invention. TABLE XIII RPMI D/F eRDF28101 28102 Glucose 2.00 3.15  3.42 6.846 10.269 mg/l Amino acids 1.041.11  3.1 6.251 15.628 mg/l Amino acids 5.6 6.6 16 24.8 41.1 (d.w. ofcomponents)

[0051] The correlation of % amino acid content in medium with MABproduction is presented in FIG. 6.

[0052] A novel cell culture media which improves protein production bycells of all types including mammalian cells which express recombinantprotein vectors has been disclosed. The invention will substantiallyenhance the cost effectiveness of cell culture procedures generallyincluding the production of monoclonal antibodies.

BIBLIOGRAPHY

[0053] (1) Murakami, H. (1989) Serum-free media used for cultivation ofhybridomas. In: A. Mizrahi(Ed.), Advances in Biotechnological Processes,Vol. 11, Monoclonal Antibodies: Production and Application. Alan R.Liss, New York, pp. 107-141.

[0054] (2) Chua, F., et al. (1994) Journal of Immunological Methods167:109-119.

[0055] (3) Chua, F., et al. (1994) Journal of Biotechnology 37:265-275.

[0056] (4) Oh, S. K. W., et al. (1995) Biotechnology and Bioengineering48:525-535.

[0057] (5) Oh, S. K. W., et al. (1996) “Flow Cytometric Studies ofOsmotically Stressed and Sodium Butyrate-Treated Hybridoma Cells” inFlow Cytometry Applications in Cell Culture, Marcel Dekker, Inc., (Eds.M. Al-Rubeai and A. N. Emery) New York, Basel, Hong Kong, pp. 101-119.

What we claim:
 1. In a cell culture medium comprising an aqueoussolution of amino acids and a carbohydrate energy source for cellscultured in said medium, the improvement wherein said aqueous solution(i) is substantially saturated with said amino acids at a temperature of30° to 50° C.; (ii) has an osmolarity of 320 to 450 mOsm; (iii) contains5.50 to 20 grams per liter of amino acids and 5.50 to 20 grams per literof said carbohydrate energy source and wherein the dry weight of theamino acids in solution in said medium comprises at least 20% of thetotal dry weight of all solid components present in said medium.
 2. Theclaim 1 cell culture medium in which said carbohydrate energy source isglucose.
 3. The claim 1 cell culture medium in which said osmolarity offrom 320 to 450 mOsm is provided at least in part by sodium salt.
 4. Theclaim 1 or claim 2 cell culture medium in which said osmolarity of from320 to 450 mOsm is provided at least in part by a sodium chlorideosmolyte.
 5. The claim 1 cell culture medium further comprising asuspension of undissolved amino acids in said aqueous solutionsubstantially saturated with amino acids wherein said substantialsaturation is maintained as cell growth consumes amino acids in solutionin said aqueous medium.
 6. In an aqueous basal cell culture medium, theimprovement which comprises providing in said basal medium (i) aconcentration of one or more amino acids reactive with cell transportsystem A to provide a total amino acid concentration of 5.50 to 20 gramsper liter; (ii) an osmolarity of from 320 to 420 mOsm  said osmolaritybeing provided by a sodium salt osmolyte.
 7. The claim 6 cell culturemedium in which the osmolyte (ii) is sodium chloride.
 8. The claim 6cell culture medium in which said one or more amino acids reactive withcell transport system A are zwitterionic amino acids.
 9. The claim 6cell culture medium in which said one or more amino acids reactive withcell transport system A are selected from the group consisting ofalanine, glycine, histidine, methionine, proline and serine.
 10. Theclaim 6 or claim 7 or claim 8 cell culture medium in which said basalcell culture medium is RPMI, DMEM, Ham's F12, RDF or eRDF.
 11. The cellculture medium 28101, 28102 or
 28103. 12. The method which comprisesculturing a cell in the culture medium of claim 1, claim 2, or claim 3,or claim
 11. 13. A method which comprises: (i) providing the cellculture medium of claim 1 or claim 2 or claim 3 or claim 11; (ii)culturing a CHO cell containing a recombinant expression vector in themedium provided in step (i); and (iii) recovering the recombinantprotein expressed by said CHO cell.
 14. The claim 13 method in which theCHO cell containing a recombinant expression vector is a dicistronicDHFR intron expression vector.
 15. A method which comprises culturing aCHO cell in culture medium
 28103. 16. The method of claim 13 whereinsaid cell cultured in said culture medium is a hybridoma.
 17. The methodwhich comprises culturing a mammalian cell in the culture medium ofclaim 1, claim 2 or claim 3 or claim
 11. 18. The method which comprisesculturing a mammalian cell having an expression vector for a recombinantprotein in a culture medium of claim 1, claim 2 or claim 3 or claim 11.19. The method which comprises culturing a CHO cell or a BHK cell or aCO5 cell or a Namaliva cell having expression vector for a recombinantprotein in a culture medium of claim 1, claim 2, claim 3 or claim 11.20. The method which comprises culturing a cell in the culture medium ofclaim 6 or claim 7 or claim 8 or claim
 9. 21. The method which comprisesculturing a mammalian cell in the culture medium of claim 7 or claim 8or claim
 9. 22. The method which comprises culturing a mammalian cellhaving an expression vector for a recombinant protein in a culturemedium of claim 7 or claim 8 or claim
 9. 23. The method which comprisesculturing a CHO cell or a BHK cell or a CO5 cell or a Namaliva cellhaving expression vector for a recombinant protein in a culture mediumof claim 7 or claim 8 or claim
 9. 24. A dry mixture of cell culturemedium components comprising amino acids, carbohydrates and vitamins,wherein said amino acids component comprise at least 20% of the dryweight of said mixture.
 25. The claim 24 mixture wherein said aminoacids component comprises from 30% to 50% of the dry weight of saidmixture.